Asprosin inhibits macrophage lipid accumulation and reduces atherosclerotic burden by up-regulating ABCA1 and ABCG1 expression via the p38/Elk-1 pathway

Background Asprosin, a newly discovered adipokine, is a C-terminal cleavage product of profibrillin. Asprosin has been reported to participate in lipid metabolism and cardiovascular disease, but its role in atherogenesis remains elusive. Methods Asprosin was overexpressed in THP-1 macrophage-derived foam cells and apoE−/− mice using the lentiviral vector. The expression of relevant molecules was determined by qRT-PCR and/or western blot. The intracellular lipid accumulation was evaluated by high-performance liquid chromatography and Oil red O staining. HE and Oil red O staining was employed to assess plaque burden in vivo. Reverse cholesterol transport (RCT) efficiency was measured using [3H]-labeled cholesterol. Results Exposure of THP-1 macrophages to oxidized low-density lipoprotein down-regulated asprosin expression. Lentivirus-mediated overexpression of asprosin promoted cholesterol efflux and inhibited lipid accumulation in THP-1 macrophage-derived foam cells. Mechanistic analysis revealed that asprosin overexpression activated p38 and stimulated the phosphorylation of ETS-like transcription factor (Elk-1) at Ser383, leading to Elk-1 nuclear translocation and the transcriptional activation of ATP binding cassette transporters A1 (ABCA1) and ABCG1. Injection of lentiviral vector expressing asprosin diminished atherosclerotic lesion area, increased plaque stability, improved plasma lipid profiles and facilitated RCT in apoE−/− mice. Asprosin overexpression also increased the phosphorylation of p38 and Elk-1 as well as up-regulated the expression of ABCA1 and ABCG1 in the aortas. Conclusion Asprosin inhibits lipid accumulation in macrophages and decreases atherosclerotic burden in apoE−/− mice by up-regulating ABCA1 and ABCG1 expression via activation of the p38/Elk-1 signaling pathway. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-022-03542-0.


Introduction
Atherosclerosis is a key etiology of cardiovascular disease, including myocardial infarction, unstable angina and ischemic stroke. Macrophages are the most abundant cell type in atherosclerotic lesions and have an essential role during all stages of this disease, from lesion initiation to ultimately rupture of arterial plaques [1]. The transformation of macrophages into lipid-rich foam cells in the subendothelial space, a hallmark of early stage atherosclerotic lesions, is mainly caused by the imbalance between modified lipoprotein uptake and cholesterol efflux [2]. ATP-binding cassette (ABC) transporters A1 (ABCA1) and ABCG1 belong to integral membrane proteins. The major function of ABCA1 is to promote the transport of intracellular free cholesterol (FC) and phospholipids to extracellular lipid-poor apolipoprotein A-I (apoA-I) for forming nascent high-density lipoprotein (HDL) particles [3]. Subsequently, ABCG1 mediates the export of FC to exogenous HDL for further maturation [4]. ABCA1-and ABCG1-dependent cholesterol efflux is the first and rate-limiting step of reverse cholesterol transport (RCT), a process by which excessive cholesterol in arterial wall cells is delivered by HDL to the liver for further metabolism and excretion [5,6]. Studies from our group and others have demonstrated that astragalin, C1q tumor necrosis factor-related protein 12, fargesin and biochanin A increase RCT efficiency and protect against atherosclerosis in apolipoprotein E-deficient (apoE −/− ) mice by up-regulating ABCA1 and ABCG1 expression [7][8][9]. Thus, searching for the novel mechanisms governing expression regulation of these two transporters is of critical importance to develop future therapeutic intervention in atherosclerotic cardiovascular disease.
Asprosin, a C-terminal cleavage product of profibrillin, contains 140 amino acid residues and is encoded by exons 65 and 66 of the Fibrillin 1 gene. As a novel glucogenic adipokine, asprosin was first discovered by Romere et al. from neonatal progeroid syndrome patients in 2016 [10]. Although white adipose tissue is regarded as a major source of asprosin, other tissues and cells such as placenta, hepatocytes and cardiomyocytes can also produce this adipokine [11,12]. Asprosin plays an important role in regulating hepatic glucose release [10], insulin secretion [13], appetite [14], and inflammatory response [15]. Of note, a recent study showed that dilated cardiomyopathy patients with lower plasma asprosin levels have an incremental risk for adverse cardiovascular events during the 5 year follow-up [16]. In patients with coronary artery disease, circulating asprosin levels are directly correlated with total cholesterol (TC) and triglycerides (TG) concentrations [17]. Additionally, injection of asprosin-pretreated mesenchymal stromal cells markedly elevates left ventricular ejection fraction and ameliorates myocardial fibrosis four weeks after myocardial infarction in mice [18]. These findings suggest an association of asprosin with lipid metabolism and cardiovascular disease. However, it is largely unknown whether and how asprosin has an effect on ABCA1 and ABCG1 expression and atherosclerosis.
Mitogen-activated protein kinases (MAPKs) are serine-threonine kinases and comprise three members in mammals: extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38. Activation of p38 has been shown to regulate macrophage lipid accumulation and plaque formation [19,20]. ETSlike transcription factor (Elk-1) belong to the ETSdomain family and can be phosphorylated and activated by MAPKs [21]. We have previously demonstrated that heat shock protein 70 decreases ABCA1 and ABCG1 expression by preventing Elk-1 from binding to their promoters in macrophages [22]. Nevertheless, very little is known about the contribution of the p38/Elk-1 signaling cascade to asprosin-regulated ABCA1 and ABCG1expression.
In this study, we found that asprosin expression was significantly down-regulated in THP-1 macrophages treated with oxidized low-density lipoprotein (ox-LDL). Importantly, lentivirus-mediated overexpression of asprosin activated the p38/Elk-1 pathway to promote ABCA1-and ABCG1-dependent cholesterol efflux in THP-1 macrophage-derived foam cells and decrease atherosclerotic plaque burden in apoE −/− mice fed a Western diet. To our knowledge, this is the first to demonstrate a link of asprosin to macrophage lipid accumulation and atherosclerosis development.

Cell culture and treatments
THP-1 cells were cultured in RPMI 1640 medium (Solarbio, Beijing, China) containing 2% penicillin-streptomycin and 10% FBS in a humidified atmosphere of 5% CO 2 at 37 °C. The differentiation of monocytes to macrophages was induced by the addition 160 nM PMA for 24 h. After 48 h of incubation with 50 µg/mL ox-LDL, macrophages were converted to foam cells. THP-1 macrophage-derived foam cells were seeded in 24-well plates (2 × 10 6 cells/well) and then transduced with empty vector (LV-Mock; Genechem) or LV-Asprosin at a multiplicity of infection of 100 in the presence of 8 mg/ mL polybrene for 24 h. Subsequently, the medium was changed to fresh 10% FBS/RPMI 1640 medium. After 48 h culture, the transfection efficiency was evaluated by western blot analysis of asprosin protein expression. To interfere with the p38/Elk-1 signaling pathway, THP-1 macrophage-derived foam cells were treated with 10 μM SB203580 for 6 h or transfected with Elk-1 S383A through lentiviral vector for 72 h. Thereafter, cells were transduced with or without LV-Asprosin for an additional 72 h.

High-performance liquid chromatography (HPLC)
The intracellular contents of TC, FC and cholesterol ester (CE) were detected using the HPLC analysis as previously described [23]. Briefly, THP-1 macrophage-derived foam cells were washed three times with phosphate-buffered saline (PBS) and then broken by an ultrasonic processor (Scientz, Zhejiang, China) on the ice. Cell lysates were centrifugated at 12,000 g for 5 min to collect protein samples, followed by detection of protein concentration using the BCA Protein Assay Kit (Beyotime). Cholesterol was extracted from protein samples by n-hexane-isopropanol (3:2, V/V) and then dissolved in isopropanol. Subsequently, 0.4 U cholesterol oxidase were added to each sample for measuring the FC content. The TC content was examined by using 0.4 U cholesterol oxidase in combination with 0.4 U cholesterol esterase. CE was calculated by subtracting FC from TC.

Oil red O staining
After transfection with LV-Mock or LV-Asprosin, THP-1 macrophage-derived foam cells were washed with PBS and then fixed in 4% paraformaldehyde solution for 10 min. Cells were stained with 0.5% Oil Red O solution (Solarbio) in the dark at 37 °C for 5 min, destained with 60% isopropanol for 15 s, incubated with hematoxylin for counterstaining, and then photographed at× 400 magnification.

Detection of NBD-cholesterol efflux
The NBD-cholesterol efflux assay was performed according to our previous report [24]. In brief, THP-1 macrophage-derived foam cells receiving the indicated treatments were incubated in phenol red-free RPMI 1640 medium supplemented with 5 µmol/L NBD-cholesterol (Invitrogen, Carlsbad, CA, USA) for 4 h at 37 °C. After washing three times with PBS, cells were incubated with 25 μg/mL apoA-I and 50 μg/mL HDL as lipid acceptors for 4 h to examine ABCA1-and ABCG1-dependent cholesterol efflux, respectively. The medium was collected, and cells were harvested and then lysed by 0.1% Triton X-100 (Gibco, Carlsbad, CA, USA). The fluorescence-labeled cholesterol in the medium and cell lysates was visualized by an inverted fluorescence microscope (Olympus IX53, Tokyo, Japan). The Image-Pro Plus software (Diagnostic Instruments, USA) was employed to analyze fluorescence intensity. NBD-cholesterol efflux capacity was calculated as the percentage of fluorescence density in the medium to total fluorescence density (medium + cell lysates).

Detection of interleukin (IL)-1β and IL-6 secretion
After the indicated treatments, cell culture supernatant was collected to detect the levels of IL-1β and IL-6 by using the commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D systems) according to the manufacturer's instructions. The absorbance at 450 nm was determined by the iMark ™ microplate reader (Bio-Rad, Hercules, CA, USA).

Measurement of Dil-ox-LDL uptake
The uptake of Dil-ox-LDL by THP-1 macrophage-derived foam cells was performed as previously described [9]. In brief, THP-1 macrophage-derived foam cells were transfected with LV-Mock or LV-Asprosin for 72 h, washed twice with PBS, and then incubated with 10 μg/mL Dilox-LDL for 4 h at 37 °C. Cells were washed three times with PBS and then visualized by an inverted fluorescence microscope.

Animals, diets and treatments
Thirty 8-week-old male apoE −/− mice with a C57BL/6 background were provided by Cavens lab animal (Jiangsu, China) and maintained under a specific-pathogen free condition. These mice were randomly divided into LV-Mock group and LV-Asprosin group, with 15 animals in each group. All mice were fed a Western diet (0.3% cholesterol, 21% fat; Research Diets) for 12 weeks to establish an atherosclerosis model. During this process, LV-Mock or LV-Asprosin (5 × 10 7 TU/mouse) were injected into the mice via the tail vein once 3 weeks. At the end of the study, the mice were weighed, anesthetized, and then euthanized to collect the blood, aortas and hearts for further analyses. The animal protocols were reviewed and approved by the Animal Care and Use Committee of the First Affiliated Hospital of University of South China.

Atherosclerotic lesion analyses
The hearts and aortic tissues were immediately isolated from the ascending aorta to the ileal bifurcation and fixed in 4% paraformaldehyde solution after apoE −/− mice were sacrificed. The adventitia was thoroughly cleaned under a dissecting microscope. The aortas were unfolded along the longitudinal axis, stained with 0.5% Oil Red O solution for 30 min and then photographed using a digital camera. The atherosclerotic burden was calculated as the percentage of Oil Red O-positive area to total aorta area. After washing with PBS, the hearts with upper aortic root were embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and then serially sectioned (8 μm thickness) throughout three aortic valves. Subsequently, the snap-frozen crosssections were stained with hematoxylin and eosin (HE), Oil Red O and Masson Trichrome for quantification of lesion area, lipid deposition and collagen content using the Image-Pro Plus 6.0 software.

Detection of plasma lipid and PCSK9 levels
Blood samples were collected from the retro-orbital plexus of apoE −/− mice at the end of experiments and then centrifuged at 800 g for 10 min to isolate the plasma. The levels of TC, low density lipoprotein cholesterol (LDL-C), HDL cholesterol (HDL-C) and TG in plasma were measured by using the commercially available enzymatic kits (Biosino, Beijing, China). Additionally, plasma PCSK9 levels were detected by using ELISA (R&D systems) according to the manufacturer's protocol.

RCT assay
The RCT assay was conducted as we had described previously [8]. J774 macrophages were loaded with 5 μCi/ mL [ 3 H]-cholesterol plus 50 μg/mL ac-LDL for 48 h and then suspended in Dulbecco's Modified Eagle Medium (Gibco). Subsequently, J774 macrophages labeled with [ 3 H]-cholesterol (4.5×10 6 cells/mouse) were intraperitoneally injected into apoE −/− mice (n = 5 in each group). Plasma samples were taken at 6, 24, and 48 h after injection. The feces were continuously collected until 48 h, and hepatic tissue specimens were obtained at 48 h. The radioactivity of [ 3 H]-cholesterol in the plasma, liver and feces was analyzed by a liquid scintillation counter. The efficiency of in vivo RCT was calculated as the percentage of radioactivity in the plasma, liver or feces to total radioactivity injected at baseline.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cultured cells and mouse aortas using a TRIzol kit (Invitrogen). The obtained RNA was converted to cDNA using a Superscript First-Strand cDNA Synthesis Kit (Invitrogen). Thereafter, qRT-PCR was performed by using SYBR Green Real-Time PCR Master Mix (Promega) on an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers used in this study were designed and synthesized by Sangon Biotech (Shanghai, China). Their sequences are presented in (Additional file 2: Table S1). The relative expression levels of target genes were determined by the 2 −ΔΔ Ct method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Statistical analysis
All results are presented as the mean ± standard deviation (SD). An unpaired two-tailed Student's t-test was applied to compare the differences between two groups. The differences among ≥ three groups were analyzed using one-way ANOVA followed by Tukey's multiple comparison test. A value of P < 0.05 was thought to be

Asprosin inhibits lipid accumulation in THP-1 macrophage-derived foam cells
Lipid accumulation in macrophages is regarded as a major driver of atherosclerosis. To clarify whether asprosin plays a role in this process, we first detected its expression using qRT-PCR and western blot in THP-1 macrophages loaded with or without ox-LDL. As shown in Fig. 1A, the mRNA and protein levels of asprosin were significantly decreased in ox-LDL-treated THP-1 macrophages compared with unstimulated cells, indicating a link between asprosin and lipid metabolism. Subsequently, THP-1 macrophage-derived foam cells were transfected with LV-Mock or LV-Asprosin to overexpress asprosin. The transfection efficiency was verified by a marked elevation in asprosin protein levels after LV-Asprosin treatment (Fig. 1B). At the same time, the HPLC analysis showed that asprosin overexpression dramatically reduced the amounts of intracellular TC, FC, and CE (Fig. 1C). Oil Red O has been widely employed to stain neutral lipids, including those present in lipid-rich foam cells in vitro and in vivo [25]. The Oil Red O staining results demonstrated that intracellular lipid droplets were significantly decreased in response to LV-asprosin transduction (Fig. 1D). These findings suggest that asprosin plays a protective role in controlling lipid accumulation within THP-1 macrophage-derived foam cells.

Asprosin increases ABCA1 and ABCG1 expression, promotes cholesterol efflux and inhibits inflammatory response in THP-1 macrophage-derived foam cells
It is well established that decreased cholesterol efflux mediated by ABCA1 and ABCG1 is a major cause leading to intracellular lipid accumulation [26]. To reveal the underlying mechanism by which asprosin protects against macrophage lipid accumulation, we examined the effect of asprosin on these two transporter expression and cholesterol efflux capacity in THP-1 macrophagederived foam cells. As expected, asprosin overexpression augmented the mRNA and protein levels of ABCA1 and ABCG1 ( Fig. 2A), with an accompanying increase in NBD-cholesterol efflux to apoA-I (Fig. 2B) and HDL (Fig. 2C). CD36 and SR-A belong to the members of scavenger receptor family and play a central role in mediating the internalization of modified lipoproteins by macrophages [27]. In addition to decreased cholesterol efflux, increased cholesterol influx contributes to intracellular lipid accumulation [28]. However, we found no significant difference in CD36 and SR-A expression in THP-1 macrophage-derived foam cells transfected with LV-Asprosin compared with LV-Mock (Fig. 2D). Consistently, no change was detected in Dil-ox-LDL uptake between LV-Asprosin group and LV-Mock group (Fig. 2E), thereby ruling out the possibility that asprosininduced prevention of macrophage lipid accumulation is caused by decreased cholesterol uptake. Collectively, these observations suggest that asprosin suppresses lipid deposition in THP-1 macrophage-derived foam cells by stimulating ABCA1-and ABCG1-dependent cholesterol export.
In addition to promoting intracellular cholesterol release, ABCA1 and ABCG1 play an important role in inhibiting inflammatory response [29,30]. The above studies have demonstrated that asprosin functions as a positive regulator of ABCA1 and ABCG1 expression. We next evaluated the impact of asprosin on pro-inflammatory cytokine production and secretion in THP-1 macrophage-derived foam cells. Transfection with LV-Asprosin dramatically down-regulated the mRNA expression of IL-1β and IL-6 (Additional file 1: Fig. S1A). Asprosin overexpression also diminished the levels of IL-1β and IL-6 in the cell culture supernatant (Additional file 1: Fig. S1B). These data suggest that asprosin contributes to alleviation of macrophage inflammatory response.

Asprosin up-regulates ABCA1 and ABCG1 expression by phosphorylating Elk-1 at Ser383
LXRα, a member of the nuclear receptor superfamily, is thought to be the most important transcription factor to induce ABCA1 and ABCG1 expression [31]. Unexpectedly, we found no difference in LXRα mRNA and protein expression in THP-1 macrophage-derived foam cells treated with LV-Asprosin compared with LV-Mock (Fig. 3A). This finding indicates that LXRα is not implicated in asprosin-enhanced ABCA1 and ABCG1 expression. Interestingly, PCSK9, a subtilisin family-serine protease predominantly expressed in the liver, has been reported to inhibit ABCA1 expression in macrophages [32]. However, asprosin overexpression had no effect on the mRNA and protein levels of PCSK9 in THP-1 macrophage-derived foam cells (Fig. 3B). Thus, like LXRα, PCSK9 is not involved in asprosin-induced up-regulation of ABCA1 and ABCG1 expression. Our previous study has identified Elk-1 as a transcription factor of ABCA1 and ABCG1 [22]. We hypothesized that the stimulatory effect of asprosin on ABCA1 and ABCG1 expression is likely mediated by Elk-1. To test this hypothesis, we measured Elk-1 expression in THP-1 macrophagederived foam cells and observed similar changes of Elk-1 mRNA and protein levels between LV-Asprosin group and LV-Mock group (Fig. 3C). Given that the transcriptional activity of Elk-1 is dependent on its translocation from the cytoplasm to the nucleus [21], We extracted cytoplasmic and nuclear proteins to determine the distribution of Elk-1 in THP-1 macrophage-derived foam cells. Our results showed that asprosin overexpression increased Elk-1 expression in the nucleus but decreased its expression in the cytoplasm (Fig. 3D), suggesting that asprosin can promote the nuclear translocation of Elk-1. The phosphorylation of Elk-1 at Ser383, Ser389 or Thr417 drives it to enter the nucleus so as to exert a transcriptional activation effect [33][34][35]. Next, we examined p-Elk-1 levels at these phosphorylated sites. Interestingly, asprosin overexpression elevated p-Elk-1 levels at Ser383 but not Ser389 and Thr417 (Fig. 3E). To determine the role of Ser383 in asprosin-induced enhancement of ABCA1 and ABCG1 expression and cholesterol efflux, we constructed a phosphorylation mutant Elk-1 S383A , which was transfected into THP-1 macrophage-derived foam cells prior to LV-Asprosin treatment. Mutation to alanine of Ser383 abrogated the effect of LV-Asprosin on ABCA1 and ABCG1 expression (Fig. 3F) as well as NBDcholesterol efflux to apoA-I (Fig. 3G) and HDL (Fig. 3H).
Taken together, these data support the concept that asprosin phosphorylates Elk-1 at Ser383 to induce transcriptional activation of ABCA1 and ABCG1 and promote subsequent cholesterol efflux.

p38 is required for asprosin-induced increase in Elk-1 phosphorylation and ABCA1 and ABCG1 expression
Elk-1 can be activated by three classes of MAPKs: ERK1/2, JNK and p38 [36,37]. We wondered whether these kinases play a role in the regulation of Elk-1 phosphorylation and ABCA1 and ABCG1 expression by asprosin. To this end, we first treated THP-1 macrophage-derived foam cells with LV-Mock or LV-Asprosin. As illustrated in Fig. 4A, asprosin overexpression markedly promoted the phosphorylation of p38 but not ERK1/2 and JNK, suggesting that asprosin functions an activator of p38. Subsequently, THP-1 macrophagederived foam cells were incubated with p38 specific inhibitor SB203580, followed by transfection with LV-Asprosin. Pretreatment with SB203580 reduced the effect of LV-asprosin on Elk-1 phosphorylation at Ser383 (Fig. 4B) and nuclear translocation (Fig. 4C). Also, the promotive effect of LV-Asprosin on ABCA1 and ABCG1 expression and NBD-cholesterol efflux was significantly reversed by SB203580 (Fig. 4D-F). These results indicate that asprosin stimulates Elk-1 phosphorylation at Ser383 and promotes ABCA1-and ABCG1-dependent cholesterol release by activating p38 in THP-1 macrophage-derived foam cells.

Asprosin mitigates atherosclerosis in apoE −/− mice
To observe the influence of asprosin on atherosclerotic lesion formation, apoE −/− mice were fed a Western diet for 12 weeks and simultaneously received tail vein injection of LV-Mock or LV-Asprosin. At the end of the study, no significant difference was found in weight gain between LV-Mock and LV-Asprosin mice (Fig. 5A). The western blot results showed that LV-Asprosin mice had a 3.84-fold increase in asprosin protein expression in the aortas as compared to controls (Fig. 5B). This finding suggests a higher transfection efficiency of LV-Asprosin in vivo. Importantly, asprosin overexpression significantly diminished the atherosclerotic plaque burden in the aortic arch regions (Fig. 5C) as well as Oil Red O-positive lesion area in whole enface aortas (Fig. 5D). Consistently, LV-Asprosin mice had a marked reduction in cross-sectional lesion area, as evidenced by HE staining  (Fig. 5F), indicating that asprosin overexpression contributes to alleviation of lesional lipid deposition. The Masson Trichrome staining further showed that collagen content within the plaques was higher in LV-Asprosin mice than LV-Mock mice (Fig. 5G), indicating that asprosin overexpression promotes the shift toward a more stable plaque phenotype. These in vivo data suggest that asprosin exerts an atheroprotective effect.

Asprosin improves plasma lipid profiles and promotes RCT in apoE −/− mice
Lipid metabolism disorder is an independent risk factor of atherosclerosis [38]. To gain mechanistical insights into the antiatherogenic action of asprosin, we detected plasma lipid levels and RCT efficiency in apoE −/− mice injected with LV-Mock or LV-Asprosin. Injection of LV-Asprosin significantly increased plasma HDL-C levels but decreased LDL-C, TC and TG levels ( Fig. 6A-D). Meanwhile, the radioactivity of [ 3 H]-cholesterol in the plasma, liver, and feces was higher in LV-Asprosin group than that in LV-Mock group (Fig. 6E), suggesting that asprosin overexpression contributes to macrophage-to-feces RCT in vivo. In addition, PCSK9 plays an important role in regulating lipid metabolism [39]. Similar to the in vitro data, there was no significant difference in plasma PCSK9 levels between LV-Asprosin group and LV-Mock group (Fig. 6F). These observations reveal that asprosin protects against atherosclerosis by improving plasma lipid profiles and promoting RCT.

Asprosin stimulates the phosphorylation of p38 and Elk-1 and up-regulates the expression of ABCA1 and ABCG1 in the aortas
Finally, we isolated the aortas from apoE −/− mice to examine the phosphorylation of p38 and Elk-1 and the expression of Elk-1, ABCA1 and ABCG1. In agreement with the in vitro results, the levels of p-p38 were significantly increased in LV-Asprosin group compared with LV-Mock group (Fig. 7A). Elk-1 mRNA and protein expression in LV-Asprosin mice were not different from LV-Mock mice (Fig. 7B). However, injection of LV-Asprosin augmented p-Elk-1 levels at Ser383 (Fig. 7C) and promoted the translocation of Elk-1 from the cytoplasm to the nucleus (Fig. 7D). Also, asprosin overexpression enhanced the mRNA and protein levels of ABCA1 and ABCG1 (Fig. 7E). These findings suggest that asprosin can activate the p38/Elk-1 signaling pathway, leading to up-regulation of ABCA1 and ABCG1 expression in vivo.

Discussion
Atherosclerosis constitutes the pathological basis of most cardiovascular disease, which has long been regarded as the major cause of morbidity and mortality in the Western world and is now the first cause of death globally [40]. Adipokines, such as leptin, adiponectin and C1q tumor necrosis factor-related protein 9, have been reported to be involved in the occurrence and development of atherosclerosis [41][42][43]. As a newly discovered adipokine in 2016, asprosin has multiple effects on central nervous system, peripheral tissues and organs [44]. It is still unclear, however, whether asprosin plays a role in atherogenesis. Here, we observed a significant decrease in asprosin mRNA and protein levels in lipid-laden macrophages. Importantly, we found that apoE −/− mice receiving LV-Asprosin injection developed less lesion area and lipid deposition as well as had increased collagen content. These in vivo data provide strong evidence to support a protective role of asprosin in development of atherosclerosis. Dyslipidemia, particularly hypercholesterolemia, is thought to be a primary risk factor for atherosclerotic cardiovascular disease. RCT functions as a major approach to eliminate excessive cholesterol from the body and is a critical mechanism for the atheroprotection of HDL [45]. A recent study showed that there is a correlation of serum asprosin levels with TC and TG concentrations in patients with coronary artery disease [17]. Our data demonstrated that overexpression of asprosin in apoE −/− mice promoted macrophage-to-feces RCT together with increased HDL-C levels and decreased LDL-C, TC and TG levels. Thus, improvement of lipid metabolism disorder is a key mechanism by which asprosin protects against atherosclerosis.
Lipid accumulation in macrophages leads to foam cell formation, a hallmark of atherosclerotic lesions throughout all stages of this disease. The formation and retention of lipid-loaded macrophages in the lesion regions exacerbates the disease and accelerates the development of vulnerable plaques [46,47]. ABCA1 mediates initial Fig. 7 Asprosin promotes p38 and Elk-1 phosphorylation and ABCA1 and ABCG1 expression in the aortas. A Protein samples were immunoblotted with antibodies against p-p38, p38 or β-actin in the aortas (n = 5 mice per group). B Measurement of aortic Elk-1 expression by qRT-PCR and western blot (n = 5 mice per group). C Detection of aortic Elk-1 phosphorylation at Ser383 using western blot (n = 5 mice per group). D Western blot analysis of aortic Elk-1 protein levels in the cytoplasm and nucleus (n = 5 mice per group). E The qRT-PCR and western blot assays of ABCA1 and ABCG1 expression in the aortas (n = 5 mice per group). The results are shown as the mean ± SD. **P < 0.01, ***P < 0.001. NS indicates not significant transport of FC to apoA-I for nascent HDL biogenesis, while ABCG1 promotes subsequent continued FC efflux to these HDL particles for further maturation [28]. In cholesterol-loaded mouse peritoneal macrophages incubated with diluted human serum, ABCA1 and ABCG1 are responsible for 50% and 20% of cholesterol efflux, respectively [48]. We have previously found that prevention of ABCA1 and ABCG1 expression by angiopoietin-1 or pregnancy-associated plasma protein-A robustly reduces cholesterol export and promotes lipid accumulation in THP-1 macrophage-derived foam cells [49,50]. Similarly, our results showed that THP-1 macrophage-derived foam cells transfected with LV-Asprosin displayed a significant increase in ABCA1 and ABCG1 expression and subsequent cholesterol efflux, followed up decreased TC, FC, and CE levels. Thus, asprosin suppresses macrophage lipid accumulation and mitigates atherosclerosis by promoting ABCA1-and ABCG1dependent cholesterol efflux. Atherosclerosis is not only a disorder of lipid metabolism dysregulation, but also a chronic inflammatory disease. Although the major function of ABCA1 and ABCG1 is to promote intracellular cholesterol efflux, there is increasing evidence that these two transporters also exert an anti-inflammatory effect [29,30]. We found that asprosin overexpression decreased the production and secretion of IL-1β and IL-6 in THP-1 macrophage-derived foam cells. This suggests that inhibition of inflammatory response may be another important mechanism underlying the atheroprotective action of asprosin.
As an ETS transcription factor, Elk-1 can modulate the expression of various genes, which are involved in the regulation of multiple biological functions, including angiogenesis, cell survival and cell differentiation [36,51,52]. Overexpression of miR-150 was shown to enhance apoptosis via silencing of Elk-1 in human umbilical vein endothelial cells [53]. We previously reported that Elk-1 can directly bind to the promoters of ABCA1 and ABCG1 for transcriptional activation [22]. Elk-1 functions as a downstream effector of p38. Several lines of evidence have demonstrated that activation of p38 is able to up-regulate the expression of ABCA1 and ABCG1 in macrophages and prostate cancer epithelial cells [54][55][56]. The transcriptional activity of Elk-1 is dependent on its phosphorylation and subsequent nuclear translocation, which is regulated by the MAPKs [21]. Moreover, the phosphorylation of Ser383 is particularly important for the transactivating effect of Elk-1, because a single mutation of this residue causes a total loss of this effect [57]. In this study, we demonstrated that asprosin overexpression in THP-1 macrophage-derived foam cells activated p38, leading to the increased phosphorylation of Elk-1 at Ser383. Phosphorylated Elk-1 translocated into the nucleus to induce ABCA1 and ABCG1 expression. We also found that blockade of the p38/Elk-1 pathway by SB203580 and mutant Elk-1 S383A significantly reversed asprosin-induced enhancement of ABCA1 and ABCG1 expression as well as cholesterol efflux capacity in THP-1 macrophage-derived foam cells. Collectively, these results support the critical role of the p38/Elk-1 signaling in the induction of ABCA1 and ABCG1 expression by asprosin and the subsequent change in cholesterol efflux. It is worth noting that asprosin has been reported to phosphorylate ERK1/2 in mouse mesenchymal stromal cells [18], and JNK in mouse insulinoma MIN6 cells [13]. However, such effects were not present in THP-1 macrophage-derived foam cells transfected with LV-Asprosin. It is thus possible that activation of MAPKs by asprosin may be cell-specific. Future research will be required to test this possibility.
In summary, the present study has demonstrated a protective role for asprosin in macrophage lipid accumulation and atherosclerosis development and revealed a novel mechanism underlying the modulation of ABCA1 and ABCG1 expression. Asprosin activates p38 and then phosphorylates Elk-1 at Ser383, thereby promoting Elk-1-dependent ABCA1 and ABCG1 transactivation for enhanced cholesterol efflux from macrophages (Fig. 8). These findings identify the regulation of macrophage asprosin as a novel approach to prevent and treat atherosclerotic cardiovascular disease. Fig. 8 A model describing the mechanism for asprosin-induced atheroprotection. Asprosin activates p38 and then phosphorylates Elk-1 at Ser383. After phosphorylation, Elk-1 enters the nucleus to stimulate ABCA1 and ABCG1 transcription. Increased ABCA1 and ABCG1 expression promotes cholesterol efflux from macrophages, leading to acceleration of RCT and alleviation of atherosclerotic plaque burden